The retinoids comprise a group of compounds including retinoic acid, retinol (vitamin A), and a series of natural and synthetic derivatives that together exert profound effects on development and differentiation in a wide variety of systems. Although early studies focused on the effects of retinoids on growth and differentiation of epithelial cells, their actions have been shown to be widespread. Many recent studies have examined the effects of these molecules on a variety of cultured neoplastic cell types, including the human promyelocytic leukemia cell line, HL60, where retinoic acid appears to be a potent inducer of granulocyte differentiation. In F9 embryonal carcinoma cells, retinoic acid will induce the differentiation of parietal endoderm, characteristic of a late mouse blastocyst. Retinoic acid also appears to play an important role in defining spatio-temporal axes in the developing avian limb and the regenerating amphibian limb.
Retinoic acid has been shown to induce the transcription of several cDNAs whose gene products have been isolated by differential screening. This observation supports the hypothesis that retinoic acid exerts its action via modulation of gene expression, in a manner analogous to the way in which steroid and thyroid hormones influence their target genes.
The ability to identify compounds which affect transcription of genes which are responsive to retinoic acid or other metabolites of vitamin A, would be of significant value, e.g., for therapeutic applications. Further, systems useful for monitoring solutions, body fluids and the like for the presence of retinoic acid, vitamin A or metabolites of the latter would be of value in various analytical biochemical applications and, potentially, medical diagnosis.
Through molecular cloning studies it has been possible to demonstrate that receptors for steroid, retinoid and thyroid hormones are all structurally related. These receptors comprise a superfamily of regulatory proteins that are capable of modulating specific gene expression in response to hormone stimulation by binding directly to cis-acting elements (Evans, Science 240, 889 (1988); Green and Chambon, Trends genet. 4, 309 (1988)). Structural comparisons and functional studies with mutant receptors have established that these molecules are composed of discrete functional domains, most notably, a DNA-binding domain that is composed typically of 66-68 amino acids (including two zinc fingers), and an associated carboxy terminal stretch of approximately 250 amino acids which comprises the ligand-binding domain (reviewed in Evans, supra).
Low-stringency hybridization has permitted the isolation and subsequent delineation of a growing list of gene products which possess the structural features of hormone receptors.
Recently, a retinoic acid dependent transcription factor, referred to as RAR-alpha (retinoic acid receptor-alpha), has been identified. Subsequently, two additional RAR-related genes have been isolated; thus there are now at least three different RAR subtypes (alpha, beta and gamma) known to exist in mice and humans. These retinoic acid receptors (RARs) share homology with the superfamily of steroid hormone and thyroid hormone receptors and have been shown to regulate specific gene expression by a similar ligand-dependent mechanism (Umesono et al., Nature 336, 262 (1988)). These RAR subtypes are expressed in distinct patterns throughout development and in the mature organism.
Other information helpful in the understanding and practice of the present invention can be found in commonly assigned, co-pending U.S. patent application Ser. Nos. 108,471, filed Oct. 20, 1987; 276,536, filed Nov. 30, 1988; 325,240, filed Mar. 17, 1989; 370,407, filed Jun. 22, 1989; and 438,757, filed Nov. 16, 1989, all of which are hereby incorporated herein by reference in their entirety.